Detection, identification and functional characterisation of plant and microbial volatile organic compounds with inhibitory activity against two plant pathogens

Volatile organic compounds (VOCs) play crucial ecological roles in interactions among organisms. For example, plant VOCs can act as a powerful deterrent of herbivore insects and pathogens or they can act as resistance inducers to stimulate plant defences. Likewise, bioactive VOCs can be emitted by beneficial microorganisms and they may potentially act as key molecules in the microbe-microbe and plant-microbe communications. However, scarce information is available concerning the role of VOCs produced by grapevine (Vitis vinifera) plants and beneficial bacteria belonging to the Lysobacter genus in defence mechanisms against two important phytopathogenic oomycetes, namely Plasmopara viticola and Phytophthora infestans, which are the causal agents of grapevine downy mildew and potato late blight, respectively. The major objectives of this PhD thesis were the detection, identification and the functional characterization of VOCs from Vitis spp. and Lysobacter spp., in order to better understand their role in plant-microbe and microbe-microbe communications and to identify new active molecules from natural origin to control phytopathogens. In particular, VOCs from resistant and susceptible grapevine genotypes were identified following P. viticola inoculation and their effect as toxic molecules against downy mildew was explored (publications 1 and 2). Likewise, VOCs produced by Lysobacter spp. were identified and characterised, in order to identify microbial VOCs able to inhibit P. infestans growth (publication 3). In order to reach these goals, a headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) and proton transfer reaction time of flight-mass spectrometry (PTR-ToF-MS) have been used. Two downy mildew resistant hybrids (SO4 and Kober 5BB) and the susceptible V. vinifera cultivar Pinot noir were analysed in vitro using PTR-ToF-MS. We found that P. viticola inoculation resulted in a significant increase monoterpene and sesquiterpene emission by resistant genotypes (SO4 and Kober 5BB) and not by the susceptible cultivar (Vitis vinifera Pinot noir; publication 1). Grapevine VOCs were further identified by HS-SPME/GC-MS using greenhouse-grown plants. The four resistant genotypes tested (BC4, Kober 5BB, SO4 and Solaris) showed significantly increased production of VOCs after P. viticola inoculation under greenhouse conditions. Conversely, no significant emission of volatile terpenes was detected from Pinot noir plants after P. viticola inoculation, suggesting that VOCs of resistant genotypes could play an important role in grapevine resistance against downy mildew. The chemical structures of P. viticola-induced VOCs were identified by retention index and the GC-MS spectrum evaluation and VOCs potentially involved in the grapevine resistance were selected according to their emission profiles. Pure compounds were tested against P. viticola by leaf disk assays and different experiments were set up, in order to elucidate the efficacy of pure VOCs both in a liquid suspension of P. viticola sporangia and after application via the gas phase. These experiments revealed six (2-phenylethanol, β-caryophyllene, β-selinene, trans-2-pentenal, 2-ethylfuran, and β-cyclocitral) and four VOCs (2-phenylethanol, trans-2-pentenal, 2-ethylfuran, and β-cyclocitral) which impaired downy mildew symptoms after direct application of liquid suspension and after treatment with VOC enriched air (without direct contact with the leaf tissue), respectively. With these results we demonstrated that VOCs produced by resistant grapevine genotypes are related to post-infection mechanisms and may contribute to grapevine resistance against P. viticola by inhibition of pathogen development (publication 2). In the second part of the PhD project, the volatilome of Lysobacter spp. was characterised for its inhibitory activity against the soil pathogen P. infestans (publication 3). The effect of VOCs emitted by Lysobacter strains was demonstrated in vitro by dual-culture assay and profiles were characterised by HS-SPME/GC-MS and PTR-ToF-MS analysis. Interestingly, the biocontrol activity and VOC profiles of Lysobacter spp. depended on the bacterial growth media. In particular, VOCs with inhibitory properties (pyrazines, pyrrole and decanal) were mainly emitted by Lysobacter type strains grown on a protein-rich medium, demonstrating the importance of the culture medium composition to optimise the biocontrol efficacy of Lysobacter spp. against plant pathogens. In summary, the presented thesis showed that both analytical chemistry techniques used (PTR-ToF-MS and HS-SPME/GC-MS) can be employed synergistically to detect and identify VOCs from different biological matrixes such as leaf tissue or bacterial cultures. The presented thesis also suggested that VOCs contribute to grapevine resistance and they can effectively be used to control economically important plant pathogens such as P. viticola. Furthermore, results generated in this work indicate that nutrient availability may affect the aggressiveness of Lysobacter spp. in the soil to maximise biocontrol efficacy against P. infestans. However, further metabolomic and transcriptomic analyses are required to investigate the VOC-mediated plant defence mechanisms and to characterize metabolic changes and VOC emissions of Lysobacter spp. grown in soil condition